We study transcriptional and translational mechanisms in nutrient control of gene expression in the yeast Saccharomyces, focusing on a regulatory system that induces genes encoding amino acid biosynthetic enzymes in response to starvation for amino acids. The transcriptional activator in this pathway, GCN4, is induced at the translational level in starved cells by phosphorylation of initiation factor 2 (eIF2) by the protein kinase (PK) GCN2. Phosphorylation of eIF2 reduces the concentration of the ternary complex (TC) containing eIF2, GTP, and initiator methionyl-tRNA, that transfers tRNAiMet to the 40S ribosome. This impedes general protein synthesis but induces GCN4 translation by a reinitiation mechanism involving small upstream open reading frames (uORFs) in the GCN4 mRNA leader. A reduction in TC levels allows 40S ribosomes scanning the GCN4 mRNA leader after translating uORF1 to bypass uORFs 2-4 and reinitiate at the GCN4 start codon instead. A similar translational control mechanism involving GCN2 and the transcription ATF4 is mobilized in mammalian cells subjected to amino acid starvation. Yeast GCN2 is activated in starved cells by binding of uncharged tRNA to a histidyl-tRNA synthetase (HisRS)-like region which functions as a sensor of amino acid availability. Activation of GCN2 also requires the GCN1-GCN20 complex that binds to the N-terminal domain (NTD) of GCN2. YIH1 contains a domain related to the GCN2-NTD and we found that YIH1 overexpression competes with the GCN2-NTD for complex formation with GCN1, reducing GCN2 activation and the derepression of GCN4 translation in starved cells. Deletion of YIH1 does not constitutively activate GCN2, indicating that YIH1 does not function generally as a negative regulator of GCN2. YIH1 resides in a 1:1 complex with G-actin and genetic depletion of actin impairs the induction of GCN4 target genes in vivo; hence, it is possible that YIH1 is released from G-actin under specific physiological conditions, eg. at the site of bud growth, to inhibit GCN2 and thereby stimulate general translation initiation. Previously, we showed that the eIF3 complex, eIF1 and eIF5 reside in a multifactor complex (MFC) with the TC and we mapped the interactions between these factors in the MFC. An eIF5 mutation that disrupts an indirect contact between the NTD of eIF3c/NIP1 and eIF2beta (tif5-7A) impairs translation in vivo in a manner partially suppressed by overexpressing the TC, and it reduces TC binding to 40S subunits in vitro. Overexpressing a CTD-less form of eIF3a/TIF32, lacking a direct binding domain for eIF2beta, also produces a slow-growth phenotype partially suppressed by overexpressing TC, and exacerbated the growth defect of the tif5-7A mutant. Thus, it appears that the two independent eIF2-eIF3 contacts in the MFC make additive contributions to the efficiency of TC recruitment. Using a technique we developed for cross-linking of initiation complexes in living cells, we showed that the overexpressing CTD-less form of TIF32 in tif5-7A cells reduces TC binding to 40S subunits in vivo. However, it does not have the expected effect of constitutively inducing GCN4 translation (Gcd- phenotype). Thus, disrupting the MFC with these mutations seems to impair a function involved in ribosomal scanning or AUG recognition and obscures the effect of reduced TC recruitment on GCN4 translation. Consistent with this last conclusion, the prt1-1 mutation in eIF2b impedes translation without reducing the abundance of 48S preinitiation complexes in vivo and, interestingly, prt1-1 impairs induction of GCN4 translation in starved cells when eIF2 is phosphorylated (Gcn- phenotype). The latter can be attributed to a delay in scanning or GTP hydrolysis by ribosomes scanning the uORF1-uORF4 interval on GCN4 mRNA. Furthermore, we identified a mutation in the NTD of eIF3c/NIP1 that increases utilization of non-AUG codons as start sites (Sui- phenotype) and was lethal in cells expressing the eIF5-G31R mutant that is hyperactive in stimulating GTP hydrolysis by the TC at AUG codons. Both effects of this NIP1 mutation were suppressed by eIF1 overexpression, as was the Sui- phenotype conferred by eIF5-G31R. Interestingly, two other NIP1-NTD mutations suppressed the Sui- phenotypes produced by the eIF1-D83G and eIF5-G31R mutations. From these and other findings, we propose that the NIP1-NTD coordinates an interaction between eIF1 and eIF5 that inhibits GTP hydrolysis at non-AUG codons. Two NIP1-NTD mutations were found to derepress GCN4 translation in a manner suppressed by overexpressing the TC, indicating that MFC formation stimulates TC recruitment to 40S ribosomes. Thus, the NIP1-NTD is required for efficient assembly of pre-initiation complexes and also regulates the selection of AUG start codons in vivo. RLI1 is an essential yeast protein closely related in sequence to two soluble, non-transporter members of the ATP-binding cassette (ABC) family of proteins that interact with ribosomes and function in translation elongation (YEF3) or translational control (GCN20). We found that affinity-tagged RLI1 co-purifies with eIF3, eIF5 and eIF2, and is associated with 40S ribosomal subunits in vivo, and that depletion of RLI1 leads to a reduced rate of translation initiation that is associated with a decrease in 40S-bound eIF2 and eIF1. Mutations of conserved residues in RLI1 expected to function in ATP hydrolysis were lethal, and one such mutation had a dominant-negative phenotype, decreasing translation initiation both in vivo and in vitro in the presence of wild-type RLI1. These findings suggest a direct role for RLI1 ATPase activity in promoting translation initiation complex assembly. RLI1-depleted cells also exhibit a deficit in free 60S ribosomal subunits and RLI1-GFP occurs in the nucleus and cytoplasm of living cells. Thus, RLI1 is a soluble ABC protein with dual functions in translation initiation and ribosome biogenesis. We showed previously that optimal transcriptional activation by GCN4 requires the coactivators SAGA, SWI/SNF, Srb mediator, CCR4/NOT complex, and RSC, and that GCN4 recruits all 5 coactivators to various target genes in living cells. We found that mutations in these coactivators reduce recruitment of TATA binding protein (TBP) and RNA Polymerase II (Pol II) by Gcn4p, implicating all five in PIC assembly at Gcn4p target genes. Recruitment of Pol II was eliminated by mutations in TBP or by deletion of the TATA box, indicating that TBP binding is a prerequisite for Pol II recruitment by Gcn4p. However, several mutations in SAGA and deletion of SRB10 had a greater impact on promoter occupancy of Pol II versus TBP, suggesting that SAGA and mediator can promote Pol II binding independently of their stimulatory effects on TBP recruitment. We found that three subunits of the Gal11/tail domain of mediator, GAL11, PGD1, MED2, form a stable subcomplex in sin4 mutant cells that can interact with GCN4 in vitro and is recruited to target genes independently of the rest of mediator by GCN4 in vivo. In addition, these tail subunits are required for high-level recruitment of the rest of mediator by GCN4. Surprisingly, high-level recruitment of the mediator tail appears to be sufficient for wild-type transcriptional activation by GCN4, implying that it may contribute directly to TBP recruitment by Srb mediator. The arginine biosynthetic gene ARG1 is repressed by the ArgR/MCM1 complex in arginine-replete cells and activated by GCN4 in cells starved for any amino acid. We found that all four subunits of the arginine repressor are recruited to ARG1 by GCN4 in cells replete with arginine but starved for isoleucine/valine. By recruiting an arginine-regulated repressor, GCN4 can precisely modulate its activation function at ARG1 according to the availability of arginine.